This invention relates to a diagnostic method for detecting cell-proliferating diseases characterized by analyzing the methylation level of cytosine residues in the region involved in the expression of cytokine receptor gene.
DNA of eukaryotes happens to be methylated at cytosine residue of 5th position [Cell, 70, 5-8 (1992), Blood, 93, 4059-4070 (1999)]. It is known that the state of methylation of genome DNA of mammalians varies with differentiation or canceration of cells. Methylation reaction is catalyzed by enzymes, DNA(cytosine-5)methyltransferase (EC 2.1.1.37) [BioEssays, 17, 139-145 (1995)]. The enzyme methylates cytosine residue of dinucleotide sequence CpG or trinucleotide sequence CpNpG [N can be anyone of A (adenine), C (cytosine), G (guanine) and T (thymine)]. Furthermore, recognizing specifically the state where only one strand of double strand is methylated, the enzyme methylates cytosine residue in the complementary chain. Recently the existence of an enzyme catalyzing de-methylation of DNA was suggested [Proc. Natl. Acad. Sci. USA, 96, 5894-5896 (1999)]. It is known that the state of methylation of DNA happens to be transmitted to progeny through reproduction (meiosis) by a mode of inheritance called imprinting [Trend in Genet. (TIG), 13, 323-329 (1997)].
Non-coding regions of some genes have a part called CpG island with an abundance of CpG sequence. The state of methylation of cytosine residue in CpG island affects the transcription/expression of the gene. Namely the less methylation results in the accelerated transcription of the gene and the more methylation results in the suppressed transcription [Trends in Genet., 13, 444-449 (1997)]. CpG island affecting gene expression frequently resides in the promoter region of the gene but a case was reported where it resided in the intron [Nature, 389, 745-749 (1997)]. As for the mechanism that DNA methylation in CpG island results in suppressed gene expression, the followings are known. Methylated CpG island is combined with a protein called MeCP2(methyl CpG binding repressor 2) and activates de-acetylation enzyme of histone. As a result neighboring chromatin structure changes into condensed form, which prohibits RNA polymerase or transcription factor from entering into promoter region, and, as a result, the transcription/expression is suppressed [J. Biochem., 125, 217-222 (1999)].
Among cell-proliferating diseases it is known that DNA methylation is involved in cancer development [Adv. Cancer Res., 72, 141-196 (1998)]. However most of the reports advocate that CpG island of a cancer suppression gene is methylated, the expression is lowered and, as a result, cells become cancerous. There has been no report that methylation of cytokine receptor gene is involved. It was also not known with cell-proliferating diseases other than cancers that methylation pattern of genome DNA is changed.
Methods to analyze the state of methylation of genome DNA are known, for example, a method using methylation sensitive restriction enzymes, a method using chemical modification by hydrazine, permanganic acids or sodium bisulfite, an immunological method using antibodies specific to methylated DNA [Nucleic Acids Res., 26, 225-2264 (1998)], and affinity column chromatography method using MBD (methyl-CpG binding domain) like MeCP2 and DGGE (denaturing gradient gel electrophoresis) method [Proc. Natl. Acad. Sci. USA, 96, 2913-2918 (1999)]. Among them the method using sodium bisulfite has been widely used [Proc. Natl. Acad. Sci. USA, 89, 1827-1831 (1992), Nucl. Acids Res., 22, 2990-2997 (1994)].
The method is based on the following principle.
When single strand DNA produced by alkali denaturation is treated by sodium bisulfite, cytosine residues are changed into uracil residues by deamination, while methylated cytosine residues remain intact. Then polymerase chain reaction (hereinafter xe2x80x9cPCRxe2x80x9d) is performed using thus treated DNA as template. The primer therefor is designed to correspond to the base sequence wherein cytosines in base sequence to be amplified are replaced by thymines. When amplification is performed using such primers, methylated cytosine residues in the original genome DNA are amplified as cytosines, while unmethylated cytosine residues are amplified as thymine residues. Thus methylation in the original genome DNA is detected by determining the base sequence of the PCR product.
Psoriasis is a chronic inflammatory skin disease and a cell-proliferating disease accompanying abnormal proliferation of epidermal cells. More than 2% of caucasian population contract it [The Lancet, 350, 349-353 (1997), The Lancet, 338, 227-230 (1991), The Lancet, 338, 231-234 (1991)]. They get it after becoming adult in most cases. The cause of psoriasis is yet to be solved. Although families with high incidence of psoriasis are known and there are reports suggesting the involvement of genetic factor, the cause itself is not yet known [Nature Getetics, 14, 231-233 (1996), Science, 264, 1141-1145 (1994), Arch. Dermatol., 130, 216-224 (1994)]. On the other hand there is a report that the expression of epidermal growth factor receptor (EGF-R) at keratinocyte is accelerated at the affected part [J. Dermatol. Sci., 16, 120-128 (1998)]. While the expression of EGF-R in keratinocyte is normally induced by stimulation with interleukin-6 (IL-6), EGF-R was expressed at keratinocyte of psoriasis irrrespective of existence or absence of IL-6 stimulation.
The promoter region of EGF-R gene contains an abundance of CpG sequence and it was shown that transcription factors bind to the region [J. Biol. Chem., 266, 1746-1753 (1991), J. Biol. Chem., 263, 5693-5699 (1988)].
The sequence of the promoter region of human EGF-R gene is disclosed in accession number M38425 of GenBank database. THE CpG sequence is especially abundant and the binding sequences of transcription actor are scattered in the region about 500 bases upstream from the translation initiation point (1114th) and the region about 800 bases downstream from the initiation point of the 1st intron in the base sequence [J. Biol. Chem., 266, 1746-1753 (1991), J. Biol. Chem., 263, 5693-5699 (1988)].
Conventional diagnosis of psoriasis has been mostly performed by long time observation by dermatologists according to the diagnostic standard (PASI: psoriasis area and severity index) described in Dermatologica, 157, 238-244 (1978), J. Dermatol. Sci., 16, 165-169 (1998) and the like. Such diagnosis needs experienced dermatologists, and long time and much work or observing tissue lesions and symptoms. Therefore a speedy, reliable and reproducible diagnostic method has been desired.
Chronic rheumatoid arthritis is one of systemic autoimmune diseases and a cell-proliferating disease accompanying abnormal proliferation and inflammation of arthrosynovial cedes [Nippon Rinsho, 57, 333-338 (1999)]. The cause of chronic rheumatoid arthritis is yet to be solved. Although families with high incidence of chronic rheumatoid arthritis are known and the correlation with genotype of human histocompatibility antigen gene HLA-DR4 has been suggested, the cause itself is not yet known. There is a report that the activity of epidermal growth factor-like receptor 2 (erbB2/HER2/neu) is increased at the affected part [Seminars in Arthritis and Rheumatism, 21, 317-329 (1992)].
The sequence of the promoter region of human epidermal growth factor-like receptor 2 (erbB2/HER2/neu) gene is disclosed in accession number Z13970 of GenBank database. The region contains an abundance of CpG sequence and transcription factor Spl is suggested to bind to the region [Mol. Cell. Biol., 7, 2597-2601 (1987), Proc. Natl. Acad. Sci. USA, 84, 4374-4378 (1987), J. Biol. Chem., 265, 4389-4393 (1990), Gene 136, 361-364 (1993), Cancer Res., 54, 4193-4199 (1994)].
Conventional diagnosis of chronic rheumatoid arthritis has been performed by long time observation by specialized medical doctors according to the diagnosis standard described in [Arthritis Reum., 31, 315-324 (1988)] and the like. Such diagnosis needs experienced medical doctors, and long time and much work for observing tissue legions and symptoms. Therefore a speedy, reliable and reproducible diagnostic method has been desired.
The invention is to provide a speedy, reliable,and reproducible method for diagnosing cell-proliferating diseases.
The invention relates to below mentioned (1)-(16).
(1) A diagnostic method for detecting cell-proliferating diseases characterized by determining the methylation level of cytosine residues at the specific region of genome DNA involved in the expression of cytokine receptor gene.
(2) A diagnostic method described in (1) wherein the cytokine receptor gene is the gene of a receptor selected from tyrosine kinase receptor family, serine-threonine kinase receptor family, interleukin receptor family, interferon receptor family, immunoglobulin receptor family, apoptotic receptor family and seven transmembrane receptor family.
(3) A diagnostic method described in (2) wherein the tyrosine kinase receptor gene is the gene of a receptor selected from epidermal growth factor receptor, epidermal growth factor-like receptor 2 (erbB2/HER2/neu), platelet derived growth factor receptor and vascular endothelial cell growth factor receptor.
(4) A diagnostic method described in (1) wherein the cell-proliferating disease is a cell-proliferating disease selected from psoriasis, chronic rheumatoid arthritis, arteriosclerosis, restenosis, diabetic retinopathy, retinopathy of prematurity and solid tumor.
(5) A diagnostic method described in (1) wherein the specific region is a region in CpG island of promoter or intron.
(6) A diagnostic method described in (1) wherein the specific region is a region involved in the expression of epidermal growth factor receptor gene and a region represented by the nucleotide sequence from 381st position to 962nd position in the nucleotide sequence as described in Seq. ID No. 4.
(7) A diagnostic method described in (6) characterized by determining the methylation level of 668th, 671st, 687th and 697th cytosine residues in the nucleotide sequence as described in Seq. ID No. 4.
(8) A diagnostic method described in (7) characterized by analyzing the methylation level of 668th cytosine residue in the nucleotide sequence as described in Seq. ID No; 4.
(9) A DNA primer having nucleotide sequence represented by Seq. ID No. 1 or 2 used for diagnostic method described in any one of (1) to (8).
(10) A diagnostic method described in (1) wherein the specific region is the region involved in the expression of epidermal growth factor-like receptor 2 (erbB2/HER2/neu) and represented by nucleotide sequence of Seq. ID No. 8.
(11) A diagnostic method described in (10) characterized by determining the methylation level of 268th, 276th and 288th cytosine residues in the nucleotide sequence as described in Seq. ID No. 8.
(12) A diagnostic method described in (11) characterized by analyzing the methylation level of 268th cytosine residue in the nucleotide sequence as described in Seq. ID No. 8.
(13) A DNA primer having nucleotide sequence represented by Seq. ID No. 5 or 6 used for any one of diagnostic methods described in (1) to (4) and (10) to (12).
(14) A DNA having nucleotide sequence represented by Seq. ID No. 1, 2, 5 or 6.
(15) A method of detecting the methylation level of cytosine residue(s) in the specific region of DNA involved in the expression of cytokine receptor gene.
(16) A method described in (15) wherein the method of detecting the methylation level is A method using methylation sensitive restriction enzyme, a method using chemical modification by hydrazine, permanganic acids or sodium bisulfite, an immunological method using antibodies specific to methylated DNA, affinity column chromatography method or DGGE (denaturing gradient gel electrophoresis) method.
This invention relates to a diagnostic method for detecting cell-proliferating diseases characterized by determining the methylation level of cytosine residues at the specific region of genome DNA involved in the expression of cytokine receptor gene.
Cytokine is a general term for protein cell-cell signal transduction molecules, which regulate proliferation or differentiation of animal cells.
Examples of cytokine receptors include tyrosine kinase receptor family, serine-threonine kinase receptor family, interleukin receptor family, interferon receptor family, immunoglobulin receptor family, apoptotic receptor family and seven transmembrane receptor family.
Examples of tyrosine kinase receptor family include epidermal growth factor-receptor (hereinafter xe2x80x9cEGF-Rxe2x80x9d) [J. Biol. Chem., 266, 1746-1753 (1991), J. Biol. Chem., 263, 5693-5699 (1988)], epidermal cell growth factor-like receptor 2 (erbB2/HER2/neu) [Biochim. Biophys. Acta, 1377, M25-M37 (1998), Biochim. Biohpys. Acta, 1198, 165-184 (1994), Molec. Cell. Biol., 7, 2597-2601 (1987)], platelet derived growth factor-receptor (hereinafter xe2x80x9cPDGF-Rxe2x80x9d) [J. Biol. Chem., 269, 32023-32026 (1994), Oncogene, 10, 1667-1672 (1995)], vascular endothelial growth factor-receptor (hereinafter xe2x80x9cVEGF-Rxe2x80x9d) [J. Biol. Chem., 270, 27948-27953 (1995)].
Examples of the region involved in the expression of the above-mentioned receptor genes include the region from xe2x88x92152nd nucleotide to xe2x88x92733rd nucleotide upstream from translation initiation point of EGF-R gene, the region from xe2x88x921st to xe2x88x92647th nucleotide upstream from translation initiation point of erbB2/HER2/neu gene [Mol. Cell. Biol., 7, 2597-2601 (1987)], the region from xe2x88x921st to xe2x88x922060th nucleotide upstream from transcription initiation point of PDGF-R gene [Oncogene, 10, 1667-1672 (1995)], the region from xe2x88x92720th to +548th nucleotide around transcription initiation point of VEGF-R gene [J. Biol. Chem., 270, 27948-27953 (1995)] and the like.
Cell-proliferating diseases are diseases like psoriasis characterized by keratin hypertrophy accompanying proliferation of epidermal cells and abnormal keratinization, chronic rheumatoid arthritis characterized by proliferation of synovial cell and villus hypertrophy, arteriosclerosis and restenosis characterized by proliferation of arterial smooth muscle cells and vascular media hypertropby, diabetic retinopathy, retinopathy of prematurity and solid tumor characterized by proliferation of vascular endothelial cells and vascularization, and the like.
The diagnostic method is described referring to psoriasis as an example in the following.
Genome DNAs of a person to be diagnosed are collected from samples such as blood, saliva, sperm, skin tiasue, tissue used for biopsy and the like respectively.
As methods or collecting genome DNAs from samples, a method described in Proc. Natl. Acad. Sci. USA, 74, 1245-1249 (1977), a method using ReadyAmp(trademark) Genomic DNA Purification System (Promega) and the like are used.
As methods for determining the methylation level of genome DNAs obtained in the above, a method using methylation sensitive restriction enzyme described in Nucleic Acids Res., 26, 2255-2264 (1998), a method using chemical modification by hydrazine, permanganic acids or sodium bisulfite, an immunological method using antibodies specific to methylated DNA, affinity column chromatography method using MBD (methyl-CpG binding domain) such as MeCP2 and DGGE (denaturing gradient gel electrophoresis) method [Proc. Natl. Acad. Sci. USA, 96, 2913-2018 (1999)] and the like are used. An example is described below.
According to Proc. Natl. Acad. Sci. USA, 89, 1827-1831 (1992), the genome DNA is treated by sodium bisulfite, by which treatment cytosine residues are deaminated into uracil residues while methylated cytosine residues remain as they are.
Primers are designed to amplify the region involved in EGF-R gene expression by polymerase chain reaction (PCR). The primers are designed by hypothesizing a nudeotide sequence where cytosine is replaced by uracil in the region to be amplified. As the region to be amplified, a part with an abundance of CpG nucleotide sequence is preferable. In EGF-R gene, the promoter region is a preferable example. The example is the sequence from 381st nucleotide to 962nd nucleotide in the nucleotide sequence as described in Seq. ID No. 4. 1114th nucleotide in the nucleotide sequence as described Seq. ID No. 4 corresponds to the translation initiation point of EGF-R, and the region from xe2x88x92152nd nucleotide to xe2x88x92733rd nucleotide upstream therefrom corresponds to the promoter region. Any primer is feasible if it is designed based on the part of nucleotide sequence to be amplified. The examples are nucleotide sequences represented by Seq. ID No. 1 and Seq. ID No. 2.
When PCR is performed with the above-mentioned primer, methylated cytosine residues are amplified as cytosines, while unmethylated cytosine residues are amplified as thymine residues. Therefore, the methylation level of cytosine residues in original genome is determined by decoding nucleotide sequence of DNA amplified by said PCR (hereinafter xe2x80x9cPCR productxe2x80x9d).
Decoding nucleotide sequence of said PCR product is carried out as follows:
Firstly said PCR product is fractionated, extracted and purified by agarose gel electrophoresis and the like described in J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning a Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989) (hereinafter xe2x80x9cMolecular Cloning 2nd Ed.xe2x80x9d).
Then nucleotide sequence determination reaction is performed by using said PCR product as template DNA, primer represented by Seq. ID No. 2, DNA polymerase and dideoxynucleotide. The nucleotide sequence determination reaction is performed by polymerizing nucleotide by DNA polymerase in the presence of dideoxynucleotide. Examples include a method of polymerizing RI-labeled nucleotide as described in Molecular Cloning 2nd Ed. and a method of polymerizing fluorescence-labeled nucleotide by Dye Terminator Cycle Sequencing Kit of Perkin Elmer and Thermal Cycler(trademark) of PerkinElmer.
As methods for decoding nucleotide sequence of the sample after the sequence determination reaction, the method by subjecting the reaction sample having incorporated RI-labeled nucleotide to denatured polyacrylamide gel electrophoresis followed by autoradiography (Molecular Cloning 2nd Ed.), or the method by subjecting the reaction sample having incorporated fluorescence-labeled nucleotide to automatic nucleotide sequence decoding apparatus like PRISM(trademark) 310 Genetic Analyzer (PE Applied Biotechnologies), and the like.
When nucleotide sequence of PCR product is decoded by the above-mentioned process, in the region of genome DNA where cytosine residue(s) is methylated, the signal(s) indicated by autoradiography or automatic nucleotide sequence decoding apparatus is only a peak(s) of cytosine, while in the region where unmethylated, the signal(s) is only a peak(s) of thymine. However where partially methylated, the signal is a mixture of cytosine and thymine.
To further investigate the methylation level, said PCR product is cloned in a suitable vector and nucleotide sequences of several clones are decoded.
Examples of cloning vectors are plasmid vectors like pUC19 and pBlueScript SK(xe2x88x92) (Stratagene), and phage vectors like M13mp19 and xcexgt11.
Cloning is performed according to a method described in Molecular Cloning 2nd Ed. PCR product is inserted and connected into a vector. The vector is transformed into host E. coli like JM109 strain and DH5 xcex1 strain, and colonies are selected. E. coli of each colony is cultivated and each cloned DNA is extracted and purified.
Nucleotide sequences of thus cloned DNAs are decoded by the method mentioned above. The sequence determination reaction is performed using each of cloned DNAs as template and the nucleotide sequence of reaction sample is decoded by autoradiography or automatic nucleotide sequence decoding apparatus.
Methylation pattern of cytosine residue(s) is analyzed after decoding nucleotide sequence. Specifically, xe2x88x92446th, xe2x88x92443rd, xe2x88x92427th and xe2x88x92417th cytosine residues (corresponding to 668th, 671st, 687th and 697th positions in the nucleotide sequence as described in Seq. ID No. 4) counted from the translation initiation point of EGF-R gene (corresponding to nucleotide number 1114th in the nucleotide sequence as described in Seq. ID No. 4) are analyzed. As understood from the result of diagnosis of 30 patients and 30 healthy people shown in Table 1 of Example 1(10), when the number of methylated residues among 4 residues is not more than 2, the person is diagnosed as psoriasis at the probability of about 83% (25/30). The probability of mistaking healthy person for a patient is about 13%(4/30). When xe2x88x92446th cytosine residue is not methylated, the person of sample is diagnosed as psoriasis at a probability of about 83%(25/30) as shown in Example 1(11). The probability of mistaking healthy person for a patient is about 33%(10/30).
Diagnosis of chronic rheumatoid arthritis is performed in a similar way as diagnosis of psoriasis descried in the above. As understood form the result of diagnosis of 9 patients and 3 healthy people shown in Table 2 of Example 2(9), when the total number of methylated residues among xe2x88x92380th, xe2x88x92372nd and xe2x88x92360th cytosine residues (corresponding to 268th, 276th and 288th positions in the nucleotide sequence as described in Seq. ID No. 8) upstream from translation initiation point of erbB2 gene is not more than 1, the person of sample is diagnosed as chronic rheumatoid arthritis. The probability of mistaking healthy person for a patient is about 33%(1/3). When xe2x88x92380th cytosine residue (corresponding to 268th position in the nucleotide sequence as described in Seq. ID No. 8) is not methylated, the person of sample is diagnosed as chronic rheumatoid arthritis at a probability of about 67%(6/9) as shown in Example 2(10).